TE antigen retrieval and immunostaining: Difference between revisions
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== Deparaffinisation == | == Deparaffinisation == | ||
* deparaffinise sections in xylene for 10min | |||
* hydration series | |||
:* 2 changes of 100% ethanol for 3 minutes each | |||
:* 95% ethanol 1min | |||
:* 80% ethanol 1min | |||
:* distilled water | |||
Note: Incubation times and repeats vary between protocols. | |||
== Tris EDTA heat-based antigen retrieval == | == Tris EDTA heat-based antigen retrieval == | ||
=== Principle === | === Principle === | ||
* aldehyde treatment ([[Polymethanal (paraformaldehyde)|paraformaldehyde]], glutaraldehyde) chemically fixes cells and tissue | |||
* [[Paraffin embedding and sectioning|paraffin-embedding]] allows very thin tissue sections to be cut | |||
* however, some antibodies don't work well after aldehyde fixation and paraffin embedding as opposed to other fixatives and cryosections | |||
* [[antigen retrieval]] methods can improve antibody binding by reversing chemical modification of epitopes or by unfolding/refolding epitopes | |||
=== Material === | === Material === | ||
'''Tris-EDTA Buffer''' | |||
final concentration: 10mM Tris base, 1mM EDTA Solution, 0.05% Tween 20, pH 9.0: | |||
* 1.21g [[Tris]] final 10mM | |||
* 0.37g [[EDTA]] final 1mM | |||
* distilled water to 1000 ml | |||
(or 100 ml to make 10x) | |||
* 0.5ml [[Tween 20]] final 0.05% (for 1x) | |||
The pH is typically around 9.0 with adjustment. Store the solution at room temperature for several weeks or at 4ºC for months. | |||
This buffer is basically a [[TE]] buffer with added Tween 20. | |||
=== Steps === | === Steps === | ||
* pre-heat steamer or water bath with container with Tris-EDTA Buffer until temperature reaches 95°C (do not boil) | |||
* immerse slides, cover with lid, incubate for 10-40 minutes (optimal incubation time depends on tissue and antibody) | |||
* remove the container and allow to cool at RT for 20 minutes | |||
* rinse sections in PBS Tween 20 for 2x2 min | |||
=== Comments === | === Comments === | ||
* Microwaves can also be used for heating but it is harder to control the temperature. Samples are easily overheated. | |||
* Containers immersed in water baths on the other hand may not reach 95ºC but the temperatures is more constant than in microwave treatments. Use a lid to increase the temperature of the buffer. | |||
== Immunostaining == | == Immunostaining == | ||
Perform blocking, primary antibody, washing, and secondary antibody incubations as normally. | |||
== See also == | |||
* [http://www.ihcworld.com/_protocols/epitope_retrieval/tris_edta.htm Tris EDTA antigen retrieval at IHCWorld] | |||
* [http://www.abcam.com/index.html?pageconfig=resource&rid=11488 Antigen retrieval page at abcam] | |||
* [http://www.millipore.com/techpublications/tech1/mcproto165 Antigen retrieval page at Millipore] | |||
* [http://books.google.com/books?q=tris%20edta%20antigen%20retrieval&hl=en Google Books search for "tris edta antigen retrieval"] | |||
[[Category:Protocol]] | |||
[[Category:Antibody]] | |||
[[Category:Microscopy]] | |||
[[Category:Protein]] |
Latest revision as of 12:43, 2 November 2009
This 2-part protocol describes antigen retrieval with Tris EDTA buffer followed by immunostaining. Antigen retrieval can be helpful for aldehyde-fixed, paraffin-embedded samples that don't give sufficient signal with a standard immunostaining protocol. Several antigen retrieval methods exist but, unfortunately, you need to determine empirically which one is most suited to your antibody.
Deparaffinisation
- deparaffinise sections in xylene for 10min
- hydration series
- 2 changes of 100% ethanol for 3 minutes each
- 95% ethanol 1min
- 80% ethanol 1min
- distilled water
Note: Incubation times and repeats vary between protocols.
Tris EDTA heat-based antigen retrieval
Principle
- aldehyde treatment (paraformaldehyde, glutaraldehyde) chemically fixes cells and tissue
- paraffin-embedding allows very thin tissue sections to be cut
- however, some antibodies don't work well after aldehyde fixation and paraffin embedding as opposed to other fixatives and cryosections
- antigen retrieval methods can improve antibody binding by reversing chemical modification of epitopes or by unfolding/refolding epitopes
Material
Tris-EDTA Buffer final concentration: 10mM Tris base, 1mM EDTA Solution, 0.05% Tween 20, pH 9.0:
(or 100 ml to make 10x)
- 0.5ml Tween 20 final 0.05% (for 1x)
The pH is typically around 9.0 with adjustment. Store the solution at room temperature for several weeks or at 4ºC for months.
This buffer is basically a TE buffer with added Tween 20.
Steps
- pre-heat steamer or water bath with container with Tris-EDTA Buffer until temperature reaches 95°C (do not boil)
- immerse slides, cover with lid, incubate for 10-40 minutes (optimal incubation time depends on tissue and antibody)
- remove the container and allow to cool at RT for 20 minutes
- rinse sections in PBS Tween 20 for 2x2 min
Comments
- Microwaves can also be used for heating but it is harder to control the temperature. Samples are easily overheated.
- Containers immersed in water baths on the other hand may not reach 95ºC but the temperatures is more constant than in microwave treatments. Use a lid to increase the temperature of the buffer.
Immunostaining
Perform blocking, primary antibody, washing, and secondary antibody incubations as normally.